1,1,3,3-tetramethyldisiloxane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Oct 2020 (experimental start date) - 23 Sep 2021 (final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

Adopted: 25 June 2018

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3,3-tetramethyldisiloxane

EC Number:

221-906-4

EC Name:

1,1,3,3-tetramethyldisiloxane

Cas Number:

3277-26-7

Molecular formula:

C4H14OSi2

IUPAC Name:

1,1,3,3-tetramethyldisiloxane

Test material form:

liquid: volatile

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan®:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS UK
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 211-269 g (males), 143-186 g (females)
- Fasting period before study: No
- Housing: Polycarbonate cages with a stainless steel mesh lid, 5 of the same sex per cage
- Diet: Teklad 2014C Diet, ad libitum
- Water: Potable public water, ad libitum
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY:
Supplier certificates of analysis provided and reviewed. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and, therefore, no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Oct 2020 To: 1-3 Feb 2021 (main), 1-2 Mar 2021 (recovery)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Directed flow nose-only chamber. Atmosphere was generated via a glass sintered vaporizer, with the the test item was supplied to the generator, via a feed line, from a plastic syringe driven at a constant rate by a syringe pump. Breathing quality air provided by in-house compressed air system. Inlet flow: 9 l/min (Groups 2 and 3), 17 l/min (Groups 1 and 4). Extract flow (drawn by in-house vacuum system): 20 l/min (Groups 2 and 3), 27 l/min (Groups 1 4). A length of tubing attached to a T-piece on the extract pipework was left open to draw in balance air. Flow meters calibrated daily, monitored continuously during exposure as part of the system checks (every 30 minutes).

Achieved stability in the syringes (20- and 50-ml) was 5 days at ambient temperature (21C).

TEST ATMOSPHERE
- Brief description of analytical method used: GC analysis
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No test item detected in the Group 1 atmosphere samples. The mean achieved atmosphere concentrations were 102, 99 and 101% of target for Groups 2, 3 and 4, respectively, with little daily variation.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hr/d, 5 d/wk to week 13, 6 d/wk for week 14

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air (analytical)

Remarks:

No test item detected in the Group 1 atmosphere samples

Doses / concentrations 2

Dose / conc.:

3.06 mg/L air (analytical)

Remarks:

3 mg/l nominal

Doses / concentrations 3

Dose / conc.:

6.94 mg/L air (analytical)

Remarks:

7 mg/l nominal

Doses / concentrations 4

Dose / conc.:

12.1 mg/L air (analytical)

Remarks:

12 mg/l nominal

No. of animals per sex per dose:

10M/10F (main), 10M/10F (recovery, groups 1 and 4)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on results from a 2 week inhalation dose range finding study in the rat. Concentrations up to 12 mg/l were well tolerated and did not cause any clinical or histopathological changes following 2 weeks of exposure. All concentrations were therefore expected to be tolerated over 13 weeks. A concentration of 12 mg/l was the maximum concentration targeted as this maintains a suitable safety margin below the lower explosive level (LEL) of 3600 ppm (21.2 mg/l).
- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight
- Rationale for selecting satellite groups: To assess recovery from any effects in Groups 1 and 4
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Animals (during acclimatization, exposure period, and recovery) were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
* On exposure days, three times daily (pre-exposure, upon return to home cage, as late as possible in working day)
* On non-exposure days during treatment period, twice daily (early in working day, as late as possible in working day)
* A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
For each animal, recorded one week before treatment commenced, on the day that treatment commenced (Day 1), and twice weekly throughout the study and before necropsy. Group mean weight changes were calculated from the weight changes of individual animals.

FOOD CONSUMPTION: Yes
Weight of food supplied to each cage, amount remaining, and an estimate of amount spilled was recorded for the week before treatment started and for each week throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of the animals (pre-treatment: all main and recovery; Week 14: all main Groups 1 and 4) were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). As no test item-related changes were observed, the examination was not extended to Groups 2 and 3 in Week 14 or to the recovery animals.

HAEMATOLOGY: Yes
* For all main animals at week 14, peripheral blood samples were collected under anaesthesia after overnight withdrawal of food and prior to dosing. As no test item-related changes were observed, the examination was not extended to the recovery animals.
* For the haematology parameters evaluated, see Table 1 below.
* The analyses were performed using Bayer Advia 120 analyser or a Stago STA Compact Max analyser.
* Blood films (prepared for all samples) were stained (Romanowsky) and examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser.

CLINICAL CHEMISTRY: Yes
* For all main animals at week 14 and all recovery animals (Groups 1 and 4) at recovery week 4, blood samples were collected under anaesthesia after overnight withdrawal of food and prior to dosing (where appropriate).
* For the clinical chemistry parameters evaluated, see Table 2 below.
* The analyses were performed using a Roche Cobas 6000 analyser.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
* For all main animals and all recovery animals (Groups 1 and 4) after termination, the right lung was used for bronchoalveolar lavage sampling (left lung was processed for histology and light microscopy).
* Cell pellet: A total and differential cell count of the BAL cells was performed using an XT-2000iV. A total and differential count (neutrophils, eosinophils, mononuclear cells (includes monocytes, macrophages) and lymphocytes) were reported as number of cells per animal and the differential cells also as a percentage of the total cell count.
* BALF supernatant: Analysis for lactate dehydrogenase and total protein was conducted using a Roche Cobas 6000.

Sacrifice and pathology:

SACRIFICE:
Animals were sacrificed via an overdose of intraperitoneal pentobarbitone sodium followed by exsanguination.

ORGAN WEIGHTS: Yes
For all main and recovery animals, the organs specified in Table 3 below were weighed at necropsy. For bilateral organs, left and right organs were weighed together, unless specified in Table 3. Organ weights presented both as absolute and as adjusted for terminal body weight.

GROSS PATHOLOGY: Yes
* All main study and recovery animals were subject to a detailed necropsy after termination. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
* For tissues fixed at necropsy, see Table 3 below. Tissues were routinely preserved in 10% Neutral Buffered Formalin. Exceptions: bone marrow smears (air dried, then fixed in methanol), testes (modified Davidson’s fluid), eyes (Davidson’s fluid). As indicated Table 3, the fixed bone marrow smears were retained, but not examined via histopathology (below).

HISTOPATHOLOGY: Yes
* For main animals in Groups 1 and 4, Table 3 below identifies the tissues subject to histopathology.
* For main animals in Groups 2 and 3 and for the recovery animals, only abnormalities were examined.
* Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with haematoxylin and eosin.

LIGHT MICROSCOPY: Yes
* For main animals in Groups 1 and 4, Table 3 below identifies the tissues examined via light microscopy.
* For main animals in Groups 2 and 3 and for the recovery animals, only abnormalities were examined.
* Findings were reported as "present" or assigned a severity grade (minimal, slight, moderate, marked or severe).

Statistics:

Summary statistics (e.g., means and standard deviations) were calculated from computer-stored individual raw data. All statistical analyses, also using the individual data, were carried out separately for males and females. The following data types were analysed at each timepoint separately: Body weight (using gains over appropriate study periods), haematology, blood chemistry, organ weights (absolute and adjusted for terminal body weight), and bronchoalveolar lavage data. Group comparisons were made between Group 1 versus Groups 2, 3, and 4. Collectively, the statistical methods used were endpoint specific and included: Bartlett’s test, F1 approximate test, William’s test, Dunnett’s test, t-tests, H1 approximate test, Shirley’s test, Steel’s test, Wilcoxon rank sum tests, Fisher’s exact test, and/or analysis of covariance. Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs during the detailed weekly physical examination or in relation to exposures.

Occasional wet fur and red staining were considered to be associated with the method of restraint during exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related but non-adverse effects on body weight gain were observed.

Lower than control mean body weight gain (statistically significant) was seen at 6.94 (females, 0.89X control) and 12.1 mg/l (males, 0.83X control; females, 0.73X control). However, these effects were considered non-adverse due to the small magnitude of changes and noted recovery. Following the 4-week recovery period, body weight gain was similar to, or slightly higher than control, for animals previously exposed to 12.1 mg/l.

The lower body weight gain at 3.06 mg/l (females) was considered incidental and not test item-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment-related but non-adverse effects on food consumption were observed.

Slightly lower than control food consumption was observed at 12.1 mg/l (both sexes), but was considered non-adverse due to the small magnitude of changes and noted recovery. Following the 4-week recovery period, an increase in food consumption was observed for animals previously exposed to 12.1 mg/l when compared with the treatment phase, although consumption remained slightly lower than the concurrent control data.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related ophthalmological findings.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes on haematology parameters in any treated group compared with control.

All differences from control, including those which attained a degree of statistical significance, were consistent with normal variation and considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on clinical biochemistry were observed.

Mean triglycerides were statistically significantly higher than control for males exposed to 12.1 mg/l (1.5X control), and higher than control for female groups exposed to 1,1,3,3-tetramethyldisiloxane (up to 1.3X control), although not exposure concentration related. These findings were considered not test item-related due to absence of a microscopic correlate, the small magnitude of changes and, in females, the lack of a exposure concentration relationship. Further, during recovery Week 4, mean triglycerides for animals previously exposed to 12.1 mg/l were within the range of the control data.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related organ weight changes in any treated group compared with control.

All differences from control were consistent with normal variation and considered incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related macroscopic observations were noted.

All macroscopic findings were considered spontaneous and/or incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related microscopic observations were noted.

All microscopic findings were considered spontaneous and/or incidental.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE:
There were no test item-related changes, for differential cell counts or for total protein and lactate dehydrogenase (LDH), in any treated group compared with control.

For differential cell counts, all other differences from control, including those which attained a degree of statistical significance, were consistent with normal variation and considered incidental.

After 14 weeks, mean total protein was lower than control for males exposed to 12.1 mg/l and higher than control for all 1,1,3,3-tetramethyldisiloxane exposed females. Mean LDH concentrations were higher than control for males exposed to 6.92 mg/l and all 1,1,3,3-tetramethyldisiloxane exposed female groups (not exposure concentration-related). These effects were considered not related to the test item due to the lack of an exposure-concentration relationship and the absence of consistent effects between the sexes. Further, following 4 weeks of recovery for the 12.1 mg/l group, mean LDH for males was lower than control, mean LDH for females was similar to control, and total protein for both sexes was similar to control.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

12.1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item-related adverse effects observed up to 12.1 mg/l

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a study performed according to OECD Test Guideline 413 and in accordance with GLP, Wistar rats were exposed via nose only inhalation to 0, 3.06, 6.94, and 12.1 mg/l (analytical; 0, 3, 7, 12 mg/l nominal) of 1,1,3,3-tetramethyldisiloxane vapour for 6 hours a day, 5 or 6 days a week for 14 weeks, with a 4-week recovery period. Clinical condition, body weight, food consumption, ophthalmic examination, haematology (peripheral blood), blood chemistry, bronchoalveolar lavage, organ weight, macropathology and histopathology investigations were undertaken. No test item-related adverse effects were observed up to 12.1 mg/l of 1,1,3,3-tetramethyldisiloxane vapour. The NOAEC is therefore concluded to be at least 12.1 mg/l 1,1,3,3-tetramethyldisiloxane vapour.